ASAIO Journal 2014
Tissue Engineering/Biomaterials
Fabrication of Porous Hydroxyapatite Scaffolds as Artificial Bone Preform and its Biocompatibility Evaluation Dong-Woo Jang,* Rose Ann Franco,* Swapan Kumar Sarkar,* and Byong-Taek Lee*
In this study, a novel porous hydroxyapatite scaffold was designed and fabricated to imitate natural bone through a multipass extrusion process. The conceptual design manifested unidirectional microchannels at the exterior part of the scaffold to facilitate rapid biomineralization and a central canal that houses the bone marrow. External and internal fissures were minimized during microwave sintering at 1,100°C. No deformation was noted, and a mechanically stable scaffold was fabricated. Detailed microstructure of the fabricated artificial bone was examined by scanning electron microscope and X-ray diffractometer, and material properties like compressive strength were evaluated. The initial biocompatibility was examined by the cell proliferation of MG-63 osteoblast-like cells using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Preliminary in vivo investigation in a rabbit model after 4 weeks and 8 weeks of implantation showed full osteointegration of the scaffold with the native tissue, and formation of bone tissue within the pore network, as examined by microcomputed tomography analyses and histological staining. Osteon-like bone microarchitecture was observed along the unidirectional channel with microblood vessels. These confirm a biomimetic regeneration model in the implanted bone scaffold, which can be used as an artificial alternative for damaged bone. ASAIO Journal 2014; 60:216–223.
artificial bone similar to natural bone structure is quite challenging. For successful fabrication of artificial bone, the internal architecture of the bone should ensure microarchitecture formation by guiding the natural regeneration process. Several reviews have been published on the general properties and design features of biodegradable and bioresorbable polymers and scaffolds for artificial bone fabrication.2–4 Bone is constantly undergoing remodeling processes. Critical-sized bone defects, which are inherently very difficult to heal by the bone remodeling process, need artificial aid to help the remodeling process cover up the defect site relatively shortly and with ease. The treatment has been addressed by autograft, allograft, xenograft, or synthetic bone graft. The former three have limitations in use due to problems of availability, collateral site damage, morbidity, disease transmission, or rejection by the body. Synthetic materials are a common option for bone defect repair. Studies have demonstrated that a porous microstructure is essential for fast osteointegration and proper bone cell ingrowth in artificial bone grafting. Therefore, scaffolds for bone tissue engineering must have porous structure with consideration of the size, shape, connectivity, and porosity of the pores.5,6 Accordingly, the design of porous implant materials with pore size larger than 150 μm appears to be suitable size for clinical bone graft applications. The design of porous implant materials with microstructure that mimics the bimodal structural configuration of bone (cortical and cancellous) and with a sufficient degree of interconnectivity5 is a unique challenge. Porous structures have inherently poor mechanical properties. Thus, a key problem in artificial bone fabrication is how to construct an osteoinducive microarchitecture with proper connectivity and biological performance without too much neglecting the mechanical strength. The conventional techniques for scaffold fabrication include fiber bonding, solvent casting, particulate leaching, membrane lamination, melt molding, polymer foaming, solid–liquid phase separation, textile technologies, and electrospinning.7–10 However, with traditional processing methods, the bionic microarchitecture could not be ensured. The logical choices of materials for artificial bone are HAp or tricalcium phosphate ceramic materials or a mixture of the two. These materials not only support favorable cell–material interaction but also promote bone ingrowth in vivo (osteoconduction).11,12 Bone remodeling is a multicellular phenomenon, which produces osteon bone mineral and also permits repair of microdamage.13 The distinctive characteristics of hard tissues in natural bone are the haversian lamellae, which are composed of columnar osteons, with the osteons distributed and connected into a concentric circle shape around a central axis. Because of this, it has been considered that producing artificial
Key Words: bioceramics, extrusion, microstructure, calcium phosphate, layered ceramics
Human cortical bone is a naturally occurring composite
material where around two thirds is composed of hydroxyapatite (HAp) nanocrystal.1 The distinctive characteristics of hard tissues in natural bone are the haversian lamellae, composed of columnar osteons, which are distributed into a concentric circle shape randomly around a central cavity containing bone marrow. Because of this complex microstructure, producing From the *Department of Biomedical Engineering and Materials, College of Medicine, Soonchunhyang University, Cheonan, Korea. Submitted for consideration May 2013; accepted for publication in revised form November 2013. Disclosure: The authors have no conflicts of interest to report. This work was supported by the Mid-career Researcher Program through the NRF grant funded by the MEST (No. 2009-0092808). Reprint Requests: Byong-Taek Lee, Department of Biomedical Engineering and Materials, College of Medicine, Soonchunhyang University, Cheonan 330–930, Korea. Email:
[email protected]. Copyright © 2014 by the American Society for Artificial Internal Organs DOI: 10.1097/MAT.0000000000000032
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HAp SCAFFOLD AS ARTIFICIAL BONE PREFORM
bone similar to natural bone is quite difficult due to the complex microstructure of natural bone. For a successful fabrication of natural bone, the internal architecture of the bone should be such that the scaffold not only ensures the formation of the characteristic microarchitecture but also guides the natural regeneration process for natural-bone-like organization. Current research mainly involves the fabrication and improvement of critical-sized defect sites by block, granular, or injectable bone substitutes. But replacing a whole bone section or using artificial bone within load-bearing region is basically different thing. Providing hierarchical architecture and at the same time ensuring the functional organization of different parts of the bone is a challenging task that needs a holistic approach for a successful design. Many have tried to fabricate microstructures that mimic human bone and contain many micropores using HAp/collagen composites.14 Porous HAp was investigated as artificial bone application15 but obtaining complex geometry of natural bone was not assured. New methods with stem cell approach are being investigated to fabricate artificial bone, but the robustness of the bone and the load-bearing ability of it are yet to achieve. A combination of sponge replica and electrospinning method addressed the unidirectional structure and cortical trabecular combined approach, but this too was devoid of significant load-bearing ability and scope for further improvement.9 We have already developed methods to prepare unidirectional, mechanically stable porous bodies with pore size suitable for bone regeneration.16 But mismatch of the thermal expansion coefficient of ZrO2 with that of HAp led to extensive cracking. Although our second attempt17 successfully controlled the structural integrity in the sintered scaffold, the presence of a bioinert phase prohibited the full biointegration. In the current study, we tried to fabricate all HAp porous bone preform that could be used to guide a biomimetic regeneration process after implantation. Detailed analysis of the microstructure and materials properties was conducted. In addition, the biocompatibility of the artificial bone with porous microstructure was investigated using in vitro and in vivo experiments. Experimental Procedure Extrusion Process for Fabricating Porous Composites An HAp nanopowder was synthesized in-house by precipitation method. Ethylene vinyl acetate copolymer (ELVAX 210A; Dupont, Wilmington, DE), carbon powder (
